Page 164 - Haematologica June

J. Celay et al.
homeostasis, lymphoid cells are equipped with a coordinat- ed network of ion channels and transporters in the plasma cell membrane that orchestrate the input and output of acid/base ions H+ and HCO − to maintain the pH within a
while showing only moderate effects on non-tumoral B lymphocytes. These data suggest that targeting AE2 repre- sents a novel therapeutic approach that may simultaneous- ly promote apoptosis of tumor cells and enhance T cell- mediated immune responses.
Methods
Peptides
A series of 24 linear peptides of 15 amino acids that potentially bind a short stretch of highly conserved amino acid sequences (NMTWAGARPT in human and NMTWATTI in mouse AE2), were designed. These conserved target sequences are within the third extracellular loop of the protein, which has been shown to play a key role in Cl-/HCO - exchange function.25,34 The design of
interactions between peptides based on the hydrophilicity/hydrophobicity and the net charge of the amino acid side chains of the involved peptides, as described.35 AE2 bind- ing peptides were synthesized by the solid phase method of Merrifield using the Fmoc alternative, as described.36 The purity of the peptides was analyzed by HPLC. To generate cycled peptides, based on the 3D structure of the p17AE2 peptide predicted using the de novo prediction server PEP-FOLD,37,38 two macrocyclic pep- tides with a head-to-tail cyclization (termed p17AE2-HT) and with a secondary amide as linker (termed p17AE2-Amide) were synthe- sized by solid phase synthesis (Wuxi AppTech, Shanghai, China). Synthesis and measurement of peptide metabolic stability proce- dures are detailed in Online Supplementary Methods.
Surface plasmon resonance
Peptide binding to the third extracellular loop of AE2 was ana- lyzed by surface plasmon resonance using the ProteOn XPR36 (Bio-Rad) optical biosensor, as described.39 Briefly, N-terminally
3i
narrow physiological range that is generally ~7.2.10–13 On the
other hand, cancer cells with a high rate of metabolic activ- ity have increased pHi while the extracellular space becomes acidified.14–17 Extracellular acidification of the tumor microenvironment suppresses the effector function of antitumor cytotoxic T cells and promotes tumor evasion.18,19 Moreover, early in vitro studies have shown that inhibition of the acid extruder Na+/H+ exchanger 1 (NHE1) in leukemic cells decreases their pHi leading to apoptosis.20,21 Accordingly, physiological pH sensors involved in the mod- ulation of acid-loading and acid-extruding mechanisms hold promise as targets in cancer therapeutics.22–24 Among the SLC4 family of HCO − transporters, the Na+-indepen-
3
dent Cl−/HCO − anion exchanger 2 (AE2, also referred to as 3
solute carrier family 4 member 2, SLC4A2) is considered a
master acid loader in many cell types.25,26 Under physiologi-
cal conditions, AE2 favors the extrusion of intracellular
load.27–29 Our group has shown that mice carrying targeted deletion of AE2 (Ae2a,b–/– mice) have lymphocytes with alterations in pHi, which eventually leads to a reduction in the number of Treg cells, among other alterations.30–33 These data prompted us to investigate the role of AE2 as a poten- tial target for tumor immunotherapy.
Here we report the generation and characterization of specific peptides targeting AE2 exchanger function. Our results show that AE2 binding peptides induced opposite effects on different T-cell subsets, promoting apoptosis in Treg cells while activating effector T-cell function. Targeting peptides also promoted apoptosis in tumor cells from differ- ent types of leukemia, lymphoma and multiple myeloma,
A
BC
3
binding peptides followed a methodology that assigns potential
HCO − in exchange for extracellular Cl−, resulting in an acid 3
Figure 1. Generation of func- tional peptides interacting specifically with the third extracellular loop of AE2 pro- tein. (A) Alignment of the third extracellular loop of murine and human AE2 and linear p17AE2, p19AE2, p20AE2 and truncated peptides. (B) p17AE2, p19AE2 and p20AE2 increased proliferation of effector T cells co-cultured with Treg cells in vitro. (C) Sensogram of surface plas- mon resonance showing p17AE2 binding to AE2 in a dose dependent manner. ***, P<0.001.
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haematologica | 2018; 103(6)